Enzymes are proteins and act as catalysts in biological reactions. Enzymes are used in molecular biotechnology or genetic engineering to manipulate biological material DNA. Enzymes like nucleases, ligases, polymerases, Modifying enzymes and topoisomerases are commonly used enzymes to do the job such as DNA manipulation.
Enzyme nucleases cut or digest the DNA molecule by breaking phosphodiester bond, which is present in the DNA molecule. Mainly there are two types of nucleases such as exonucleases and endonucleases. Exonucleases digest the phosphodiester bond which is present at the ends of the DNA molecule. Endonucleases digest the phosphodiester bonds which are present in the middle of the DNA molecule.
Restriction endonuclease enzymes cleave the specific phosphodiester bond present in the DNA molecule. There are three types of restriction enzymes are there, such as Type-I, Type-II and Type-III. Restriction enzyme Type-II is used commonly in molecular biotechnology or genetic engineering as they have the ability to digest phosphodiester bond present in the specific sequence of the DNA. They are code specific. To do their function they require specific temperature and magnesium concentration. They produce both blunt end and sticky end after the digestion.
Enzyme ligases help in binding two pieces of DNA by forming phosphodiester bonds. Ligases form two phosphodiester bonds one at each strand of the DNA molecule or in other words it act as biological glue. Ligases enzyme has the capacity to bind both blunt end and sticky end produced by restriction endonuclease enzymes. Adaptors, linkers and homopolymer tailing produce sticky ends.
Polymerases synthesize a new strand of DNA molecule complementary to the existing DNA or RNA strand, which acts as template. DNA polymerases require a primer to do their job that is DNA strand synthesis. DNA polymerase type-I has got both activity like DNA polymerization and also DNA degradation or nick formation. Klenow fragments are fragments of DNA polymerase type-I enzyme which has got polymerase activity but lacks the nucleases activity.
DNA Modifying Enzymes:
Enzyme alkaline phosphatase extracted from E. coli or calf intestine degrades phosphate group present in the 3 prime end of the DNA molecule.
Poly Nucleotide Kinase:
Polynucleotide kinase enzyme extracted from E. coli which is infected with bacteriophage has the capacity to add phosphate group in the 5 prime end of the DNA molecule.
Terminal deoxy-nucleotidyle transferase:
Terminal deoxy-nucleotidyle transferase enzyme is extracted from calf thymous tissue, which has got the capacity to add one or more number of nucleotide in 3 prime terminus of the DNA molecule.
Enzyme topoisomerasees change the confirmation of covalently closed circular DNA molecule, such as plasmids by removing the supercoils present in the circular DNA molecule. Enzyme topoisomerase plays a very important role during DNA replication but its use in genetic engineering is still not very clear.
All these enzymes such as endonucleases, ligases, topoisomerases, DNA or RNA polymerases are used in molecular biotechnology methods, such as genetic engineering, DNA fingerprinting, gene manipulation, gene identification and much more. Without these enzymes biotechnology cannot be used as a tool to improve the quality or quantity of the products produced by plants or genetically modified plants. All these enzymes are heart of the biotechnology applications.
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